From flies to humans, alternative splicing of pre-mRNAs is the most important molecular mechanism used to expand protein diversity1,2. The nervous system boasts the greatest complexity and diversity of alternatively spliced transcripts3. Because alternatively spliced transcription factors can regulate cell fate and neuronal wiring decisions, they are important for creating diverse cell types in the nervous system1. In Drosophila, longitudinals lacking (lola) encodes a critical transcription factor that guides CNS development by driving a range of cellular differentiation and axon guidance decisions4-9. lola undergoes complex alternative cis-splicing to make distinct isoforms. However mRNAs can also be made by inter-allelic trans-splicing, an evolutionarily conserved but poorly understood process where exons from pre-mRNAs synthesized from homologous alleles can be spliced together, resulting in transcripts that are a blend of the two parental alleles10. Since the parentl alleles are often highly similar, the blended transcripts would probably not be functionally distinct, so the function of this unique type of splicing is unknown. One novel possibility is that trans-splicing helps regulate the expression pattern of lola isoforms. My goal for the proposed research is to build a comprehensive picture of how alternative inter-allelic trans-splicing of lol produces functional complexity in the brain. To do this I first plan to map the expression of the alternatively spliced isoforms within the D. melanogaster central nervous system. In Aim 1, I will use high-throughput sequencing together with an isoform-specific reporter system to map the expression pattern of each lola isoform down to the single cell level. I will also map the expression of inter-allelic trans-spliced lola isoforms to determine whether inter-allelic trans-splicing is also spatially regulated and correlated with isoform expression patterns. In Aim 2, I plan to assess the functional role of each isoform. I will use an isoform-specific ChIP-seq analysis to identify genes targeted by each isoform. I will then make targeted mutations of variable exons in the endogenous locus and determine whether neuronal development and global mRNA expression patterns are disrupted in flies that can no longer express a particular isoform. Taken together, results from these experiments will reveal how complex alternative trans-splicing of lola directs important steps in neuronal development and wiring.